Dismantling of necrotroph Alternaria alternata by cellular intervention of Peppermint Oil Nanoemulsion (PNE).

Alternaria alternata, a common necrotrophic fungal pathogen, poses a significant threat to various crops, causing substantial yield losses and quality deterioration. In the present study, we explore the potential fungicidal properties of Peppermint Oil Nanoemulsion (PNE) against A. alternata and investigate its impact on the fungal phenotype. Our previous study synthesized the PNE using a nanoemulsion approach, optimizing its formulation for enhanced stability and efficacy. The present study extended the assessment of a multidisciplinary approach to comprehensively analyze the fungicidal efficacy of PNE against A. alternata. Notably, in a liquid growth medium, 0.5 % of PNE could reduce A. alternata's biomass by 96 %. PNE-treated mycelia were stained with a nitro-blue tetrazolium (NBT) dye to assess ROS accumulation during oxidative stress induced by PNE. A higher degree of ROS generative potential of PNE has appeared in 72 h treated mycelia. PNE-treated mycelium showed cell wall alterations, with red fluorescence peaking at 0.5 %, indicating a dose-dependent effect compared to the untreated control. Consequently, PNE treatment led to a significant early hour increase in electrical conductivity (EC), extended to 306.03-353.33 µS/cm compared to 277.67-280.33 µS/cm untreated control. Scanning Electron Microscopy (SEM) analysis of A. alternata reflects the osmotic imbalance and structural damage in mycelia as the obvious cause of fungal inhibition. In addition, a phenotype microarray analysis of PNE-treated A. alternata mycelia revealed a significant phenotypic loss in 37 out of 708 substrates, potentially impacting metabolic pathways essential for fungi's functional processes. The study found that downregulation of genes like Cre A, NmrA, SOD, IMP, EfP, and Erg, which are linked to A. alternata's stress coping mechanisms, leads to alterations in survival and adaptation. Additionally, understanding the phenotypic changes induced by PNE contributes to our knowledge of the mode of action of this nanoemulsion against A. alternata. In conclusion, this study provides a comprehensive analysis of the fungicidal and phenotypic effects of PNE, offering a promising avenue for sustainable fungal control. The implications of our research extend to the development of novel, natural fungicidal agents for agricultural applications.

Upregulation of Profilin 2 on HDAC6 overexpression in mouse GC-1 cells and its putative role in germ cell migration in the testis.

Previous reports from this laboratory have demonstrated the involvement of histone deacetylase 6 (HDAC6) in sperm motility. As the presence of HDAC6 has also been reported in the earlier stage germ cells, studies were undertaken to explore its role during these stages of spermatogenesis. HDAC6 was overexpressed in GC-1spg cells, which represent the stage between type B spermatogonia and primary spermatocyte, and its effect on germ cell transcriptome was investigated by microarray. Among the many transcripts that were differentially regulated, Profilin 2, reported previously as a neuronal specific isoform, was observed as one of the genes highly upregulated at the transcript level, which was further confirmed by real-time PCR, and the protein confirmed by indirect immunofluorescence (IIF). Profilin 2 colocalized with HDAC6, as seen both in GC-1 cells and sperm. On the sperm, the presence of Profilin 2 was detected throughout the flagella with its colocalization with HDAC6 seen conspicuously in the mid-piece region of the flagella. Co-immunoprecipitation studies confirmed Profilin 2 interaction with HDAC6. Docking studies using Z dock suggested the interaction of 8 residues of HDAC6 with 6 residues of Profilin 2. The novel observation of Profilin 2 in spermatogonial cells, its significant upregulation on HDAC6 overexpression and its interaction with HDAC6 suggests that HDAC6 in collaboration with Profilin 2 may play a role in regulating the movement of germ cells from one stage/compartment to the next.

Bacillus subtilis suppresses the charcoal rot disease by inducing defence responses and physiological attributes in soybean.

Endophytes can induce the defence responses and modulates physiological attributes in host plants during pathogen attacks. In the present study, 127 bacterial endophytes (BEs) were isolated from different parts of healthy soybean plant. Among them, two BEs (M-2 and M-4) resulted a significant antagonistic property against Macrophomina phaseolina, causes charcoal rot disease in soybean. The antagonistic potential was evaluated through dual culture plate assay, where M-4 expressed higher antifungal activity than M-2 against M. phaseolina. The M-4 produces cell wall degrading enzymes viz. cellulase (145.71 ± 1.34 µgmL-1), chitinase (0.168 ± 0.0009 unitmL-1) and ß,1-3 endoglucanase (162.14 ± 2.5 µgmL-1), which helps in cell wall disintegration of pathogens. Additionally, M-4 also can produce siderophores, indole-3-acetic acid (IAA) (17.03 ± 1.10 µgmL-1) and had a phosphate solubilization potential (19.89 ± 0.26 µgmL-1). Further, GC-MS profiling of M-4 has been carried out to demonstrate the production of lipophilic secondary metabolites which efficiently suppress the M. phaseolina defensive compounds under co-culture conditions. Bio-efficacy study of M-4 strain shown a significant reduction in disease incidence around 60 and 80% in resistant and susceptible varieties of soybean, respectively. The inoculation of M-4 potentially enhances the physiological attributes and triggers various defence responsive enzymes viz. superoxide dismutase (SOD), phenol peroxidase (PPO), peroxidase (PO) and catalase (CAT). The histopathological study also confirmed that M-4 can reduce the persistence of microsclerotia in root and shoot tissue. Conclusively, M-4 revealed as an efficient biocontrol agent that can uses multifaceted measures for charcoal rot disease management, by suppress the M. phaseolina infection and enhance the physiological attributes of soybean.

Urea hydrogen peroxide-initiated synthesis of pyranopyrazoles through oxidative coupling under base- and metal-free conditions by physical grinding method.

A novel multicomponent one-pot expeditious synthesis of highly functionalized and pharmaceutically fascinated pyranopyrazoles has been developed. This reaction occurs via tandem Knoevenagel condensation reaction of methyl aryl derivatives, 3-methyl pyrazolone and malononitrile in the presence of urea hydrogen peroxide under the physical grinding method. The present methodology offers several benefits such as available green and cheap starting materials, solvent-free, mild reaction conditions, high atom economy, eco-friendly standards, excellent yields and easy isolation of the products without column chromatographic separation.

Analysis of a novel coronavirus (2019-nCOV) system with variable Caputo-Fabrizio fractional order.

The main aim of this study is to present a new variable fractional-order derivatives for novel coronavirus (2019-nCOV) system with the variable Caputo-Fabrizio in Caputo sense. By using the fixed point theory, we explore the new existence and uniqueness results of the solution for the proposed 2019-nCOV system. The existence result is obtained with the aid of the Krasnoselskii fixed point theorem while the uniqueness of the solution has been investigated by utilizing the Banach fixed point theorem. Furthermore, we study the generalized Hyers-Ulam stability as well as the generalized Hyers-Ulam-Rassias stability and also discuss some more interesting results for the proposed system.

Harnessing Potential of ω-3 Polyunsaturated Fatty Acid with Nanotechnology for Enhanced Breast Cancer Therapy: A Comprehensive Investigation into ALA-Based Liposomal PTX Delivery.

Our hypothesis posited that incorporating alpha-linolenic acid (ALA) into liposomes containing Paclitaxel (PTX) could augment cellular uptake, decrease the therapeutic dosage, and alleviate PTX-related side effects. Our investigation encompassed characterization of the liposomal formulation, encompassing aspects like particle size, surface morphology, chemical structure, drug release kinetics, and stability. Compatibility studies were performed through Fourier transform infrared spectroscopy (FTIR). By utilizing the Box-Behnken design (BBD), we developed ALA-based liposomes with satisfactory particle size and entrapment efficiency. It is noteworthy that ALA incorporation led to a slight increase in particle size but did not notably affect drug entrapment. In vitro drug release assessments unveiled a sustained release pattern, with ALA-PTX liposomes demonstrating release profiles comparable to PTX liposomes. Morphological examinations confirmed the spherical structure of the liposomes, indicating that substituting ALA with phosphatidylcholine did not alter the physicochemical properties. Cellular uptake investigations showcased enhanced uptake of ALA-based liposomes in contrast to PTX liposomes, likely attributed to the heightened fluidity conferred by ALA. Efficacy against MCF-7 cells demonstrated concentration-dependent reductions in cell viability, with ALA-PTX liposomes exhibiting the lowest IC50 value. Morphological analysis confirmed apoptotic changes in cells treated with all formulations, with ALA-PTX liposomes eliciting more pronounced changes, indicative of enhanced anticancer efficacy.

High-altitude and low-altitude adapted chicken gut-microbes have different functional diversity.

Recently, there has been considerable interest in the functions of gut microbiota in broiler chickens in relation to their use as feed additives. However, the gut-microbiota of chickens reared at different altitudes are not well documented for their potential role in adapting to prevailing conditions and functional changes. In this context, the present study investigates the functional diversity of gut-microbes in high-altitude (HACh) and low-altitude adapted chickens (LACh), assessing their substrate utilization profile through Biolog Ecoplates technology. This will help in the identification of potential microbes or their synthesized metabolites, which could be beneficial for the host or industrial applications. Results revealed that among the 31 different types of studied substrates, only polymers, carbohydrates, carboxylic acids, and amine-based substrates utilization varied significantly (p < 0.05) among the chickens reared at two different altitudes where gut-microbes of LACh utilized a broad range of substrates than the HACh. Further, diversity indices (Shannon and MacIntosh) analysis in LACh samples showed significant (p < 0.05) higher richness and evenness of microbes as compared to the HACh samples. However, no significant difference was observed in the Simpson diversity index in gut microbes of lowversus high-altitude chickens. In addition, the Principal Component Analysis elucidated variation in substrate preferences of gut-microbes, where 13 and 8 carbon substrates were found to constitute PC1 and PC2, respectively, where γ-aminobutyric acid, D-glucosaminic acid, i-erythritol and tween 40 were the most relevant substrates that had a major effect on PC1, however, alpha-ketobutyric acid and glycyl-L-glutamic acid affected PC2. Hence, this study concludes that the gut-microbes of high and low-altitudes adapted chickens use different carbon substrates so that they could play a vital role in the health and immunity of an animal host based on their geographical location. Consequently, this study substantiates the difference in the substrate utilization and functional diversity of the microbial flora in chickens reared at high and low altitudes due to altitudinal changes.

Significance of antibody numbering systems in the development of antibody engineering.

Immunotherapy has become increasingly popular in recent years for treating a variety of diseases including inflammatory, neurological, oncological, and auto-immune disorders. The significant interest in antibody development is due to the high binding affinity and specificity of an antibody against a specific antigen. Recent advances in antibody engineering have provided a different view on how to engineer antibodies in silico for therapeutic and diagnostic applications. In order to improve the clinical utility of therapeutic antibodies, it is of paramount importance to understand the various molecular properties which impact antigen targeting and its potency. In antibody engineering, antibody numbering (AbN) systems play an important role to identify the complementarity determining regions (CDRs) and the framework regions (FR). Hence, it is crucial to accurately define and understand the CDR, FR and the crucial residues of heavy and light chains that aid in the binding of the antibody to the antigenic site. Detailed understanding of amino acids positions are useful for modifying the binding affinity, specificity, physicochemical features, and half-life of an antibody. In this review, we have summarized the different antibody numbering systems that are widely used in antibody engineering and highlighted their significance. Here, we have systematically explored and mentioned the various tools and servers that harness different AbN systems.

On the existence and stability of fuzzy CF variable fractional differential equation for COVID-19 epidemic.

In this paper, we convert the recent COVID-19 model with the use of the most influential theories, such as variable fractional calculus and fuzzy theory. We propose the fuzzy variable fractional differential equation for the COVID-19 model in which the variable fractional-order derivative is described using the Caputo-Fabrizio in the Caputo sense. Furthermore, we provide the results on the existence and uniqueness using Lipschitz conditions. Also, discuss the stability analysis of the present new COVID-19 model by employing Hyers-Ulam stability.

Revisiting the Characteristics of Testicular Germ Cell Lines GC-1(spg) and GC-2(spd)ts.

Spermatogenesis is a multifaceted and meticulously orchestrated process involving meiosis, chromatin build up, transcriptional and translational hushing, and spermiogenesis. Male germ cell lines GC-1spg (GC-1) and GC-2(spd)ts (GC-2) provide a useful resource to comprehend the molecular events occurring during such a tightly regulated process. Using cDNA microarray, expression profiling of GC-1 and GC-2 cell lines was done to precisely understand their characteristics and uniqueness. Our observations indicate that whilst both the cell lines are indeed of testicular origin, GC-2 is not haploid as was originally thought. Data analysis of the 23,351 transcripts detected in GC-1 and 20,992 in GC-2 cell lines demonstrates an 80% transcript overlap between GC-1 and GC-2 cells and ~ 40% similarity of both with the primary spermatocyte transcriptome. 3152 and 793 transcripts exclusive to GC-1 and GC-2, respectively, were identified. The presence of transcripts for 36 genes was validated in these cell lines including those showing testis-specific expression, as well as genes not reported previously. Overall, this study provides the transcriptome database of GC-1 and GC-2 cells. Analysis of the data demonstrates the transcriptomic transitions between GC-1 and GC-2 thus providing a glimpse to the process of germ cell differentiation from type B spermatogonium into preleptotene spermatocyte.

Tetrahydropyrazolopyridines as antifriction and antiwear agents: experimental and DFT calculations.

Some tetrahydropyrazolopyridines (THPP-H) with the methoxy (THPP-OMe) and methyl (THPP-Me) substituents were synthesized by a one-pot multi-component reaction. NMR spectroscopy (1H and 13C) was used to authenticate the synthesis. According to the results of tribological tests ASTM D4172, and ASTM D5183 on a four-ball tester in paraffin oil (PO) at a concentration of 0.25% w/v, their relative tribo-activity along with a reference additive, zinc dialkyldithiophosphate (ZDDP) could be figured out as mentioned below-THPP-OMe > THPP-Me > THPP-H > ZDDP. The calculation of frictional power loss from the coefficient of friction data of the tested additives supports the given order. As is apparent from AFM and SEM micrographs of the wear scar surface for plain oil with and without different tetrahydropyrazopyridines, surface evenness endorses the above trend. Proof for strong adsorption of the synthesized additives is provided by EDX analysis of the steel ball surface after performing the tribological test, where nitrogen and oxygen are vividly seen as heteroatoms. XPS studies reveal the composition of the in situ formed tribofilm. The moieties containing carbon bonded to oxygen/nitrogen as decomposed products of the additive together with oxides of iron in +II or +III oxidation states are perceptible in the tribofilm, the tribofilm interferes with the proximity of the surfaces keeping them far apart. Consequently, friction and wear are remarkably reduced. Findings from Density Functional Theory (DFT) calculations are in full agreement with the results obtained from tribological experiments.

Supplementation of Trichoderma improves the alteration of nutrient allocation and transporter genes expression in rice under nutrient deficiencies.

Nutrients are the finite natural resources that are essential for productivity and development of rice and its deficiency causes compromised yield along with reduced immunity against several biotic and abiotic stresses. In this study, the potential of Trichoderma reesei has been investigated as a biofertilizer (BF) to ameliorate nutrient stress in different rice cultivars at physiological, biochemical and molecular levels. The results indicated that cultivar Heena is much more compatible with BF as compared to cultivar Kiran at 50% nutrient limiting condition. Enhancement in physiological attributes and photosynthetic pigments were observed in BF treated Heena seedlings. The localization of biofertilizer in treated roots was further validated by scanning electron micrographs. This result correlated well with the higher levels of Indole acetic acid and Gibberellic acid in biofertilizer treated rice. Similarly, the uptake of micro-nutrients such as Fe, Co, Cu and Mo was found to be 1.4-1.9 fold higher respectively in BF treated Heena seedlings under 50% nutrient deficient condition. Furthermore, different stress ameliorating enzymes Guaiacol peroxidase, Super oxide dismutase, Total Phenolic Content, Phenol Peroxidase, Phenylalanine ammonia lyase and Ascorbate peroxidase in Heena seedlings were also increased by 1.8, 1.4, 1.2, 2.4, 1.2, and 8.3-fold respectively, at 50% nutrient deficient condition. The up-regulation of different micro and macro-nutrients allocation and accumulation; metal tolerance related; auxin synthesis genes in BF treated Heena as compared to 50% nutrient deficient condition was further supported by our findings that the application of biofertilizer efficiently ameliorated the deficiency of nutrients in rice.

Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico.

We had previously reported presence of histone deacetylase 6 (HDAC6) in sperm and demonstrated its tubulin deacetylase activity and role in sperm motility in rat. In the present study we report its abundant expression in testis, epididymis, accessory sex organs, brain, and adrenal. In the testis, HDAC6 transcript and protein were observed throughout development. We therefore cloned the gene from rat testis using primers for hdac6 (accession number XM_228753.8) in order to determine the role of acetylation/deacetylation in spermatogenesis. The cloned rat hdac6 gene is ~3.5 kb with 28 exons and 1152 amino acids. We noted 4 single nucleotide polymorphisms (SNPs) on exons 2 (G/A), 5 (A/G), 7 (T/C), and 26 (G/T), respectively, in this sequence when compared to XM_228753.8. These were further validated at both cDNA and gene level. These SNPs resulted in 2 amino acids changes, namely, glycine → arginine and valine → phenylalanine at protein level. Cloned hdac6 overexpressed in HEK293T cells demonstrated significant overexpression by IIF. Alpha-tubulin acetylation analysis of the overexpressed cell lysate demonstrated that the protein was bioactive. This is the first study showing the ontogenic expression in the testis and reporting experimentally validated sequence of rat HDAC6 and its structural and functional annotation in silico. This sequence has been submitted to GenBank (Accession number Rattus KY009929.1).

TRPV4-mediated channelopathies.

Transient receptor potential vanilloid sub type 4 (TRPV4) is a member of non-selective cation channel that is important for sensation of several physical and chemical stimuli and also involved in multiple physiological functions. Recently it gained immense medical and clinical interest as several independent studies have demonstrated that mutations in the TRPV4 gene can results in genetic disorders like Brachyolmia, Charcot-Marie-Tooth disease type 2C, Spinal Muscular Atrophy and Hereditary Motor and Sensory Neuropathy type 2. Close analysis of the data obtained from these naturally occurring as well as other TRPV4 mutants suggest that it is not the altered channel activity of these mutants per se, but the involvement and interaction of other factors that seem to modulate oligomerization, trafficking and degradation of TRPV4 channels. Also, these factors can either enhance or reduce the activity of TRPV4. In addition, there are some potential signaling events that can also be involved in these genetic disorders. In this review, we analyzed how and what extent certain cellular and molecular functions like oligomerization, surface expression, ubiquitination and functional interactions might be affected by these mutations.